Nonalcoholic fatty liver disease (NAFLD) is marked by accumulation of fat in liver cells with accompanying inflammation and variable degrees of cell injury and fibrosis. When cell injury and fibrosis are present, the disease has a potential to progress and is referred to as nonalcoholic steatohepatitis (NASH), which can lead to cirrhosis, liver cancer, morbidity and mortality. The etiology of NASH is not clear nor is there an approved treatment modality for it. NAFLD has become an extremely common disorder, estimated to affect up to 30% of individuals in the US. Unfortunately, it commonly goes unrecognized, as demonstrated by our recent finding that over a 5-year period, 28% of subjects enrolled as healthy volunteers to studies at the NIH Clinical Center were likely to have underlying NAFLD. As such, there is a clear need to understand the pathophysiology of the disease and its treatment. Our focus on NAFLD is three-fold: first, we aim to identify and refine effective treatments for the disorder. Secondly, we aim to identify and characterize key genes that play a role in the pathogenesis of NAFLD through the use of genetic studies and cell- or animal models. Our third focus is on the physiology of fat accumulation and injury in the liver, especially as it relates to handling of oral caloric load. Vitamin E has been shown in a randomized placebo-controlled trial to be an effective therapy for NASH. Curiously, treatment with vitamin E resulted not only in a decrease in injury (thought to reflect its antioxidant effect) but was also associated with a decrease in liver fat, through an unknown mechanism. Furthermore, the optimal dose of vitamin E to treat NASH is currently unknown. We conducted a clinical mechanistic trial to determine the mechanism of action of vitamin E and its optimal dose and are currently analyzing data from this study and complementary in vitro models to map the pathways affected by vitamin E and whether they are dependent on its antioxidant capacity. Genome wide association (GWA) studies identified single nucleotide polymorphisms (SNPs) that are associated with increased hepatic fat or elevated liver enzymes, presumably reflecting nonalcoholic fatty liver disease (NAFLD). We initiated a study to investigate whether these SNPs are associated with histological severity in a large cohort of NAFLD patients. 1117 (894 adults/223 children) individuals enrolled in NASH-Clinical Research Network and National Institutes of Health Clinical Center studies with histologically-confirmed NAFLD were genotyped for SNPs that are associated with hepatic fat or liver enzymes in GWA studies. We confirmed the association of the rs738409G allele in the PNPLA3 gene with steatosis and were first to describe its association with histological severity. In pediatric patients, the high-risk rs738409G allele was associated with an earlier presentation of disease. We also described a hitherto unknown association between SNPs at a chromosome 10 locus and the severity of NASH fibrosis. Similarly, we demonstrated associations of SNPs near or in the genes for hydroxysteroid (17) dehydrogenase 13 (HSD17B13), RAR-related orphan receptor (RORA) and protein phosphatase 1,regulatory subunit 3B (PPP1R3B) with histological features of NAFLD, after adjustment for age, gender and BMI. In-depth genotyping near RORA, a nuclear receptor involved in control of circadian rhythm and metabolic functions, showed that SNPs that are associated with NAFLD are located in the putative promoter region of 2 of the 4 splice variants (variants 2 and 3) as opposed to SNPs upstream of other variants, suggesting that alternation in the relative expression of the different isoforms affects fat accumulation in the liver. RORA knock-out in cell lines did not affect the degree of hepatic lipid accumulation under standard conditions. However, when cells were overloaded with nutrients (high glucose, high-fatty acids medium), the amount of intracellular fat significantly decreased with knock-down, predominantly through a decrease in the average size of the lipid droplets. A similar effect was seen in adipocytes. We have established a murine liver-specific Rora knock-out and through that demonstrate the important role of RORA on the generation of liver fat. Little is known about HSD17B13. We found it to be predominantly expressed in the liver and to colocalize with lipid droplets, but its substrate and physiological roles are unknown. In-depth genotyping of the gene region demonstrated associations of coding and splice-site SNPs in the gene with NAFLD, confirming a possible role for this enzyme in the pathogenesis of NASH. We identified a role for HSD17B13 as a key enzyme in retinoid metabolism and determined that genetic variants that are associated with NAFLD are also essential to its enzymatic activity. We are currently studying the effects of HSD17B13 in a knock-out animal model. As NAFLD is intricately related to food intake and energy metabolism, we are undertaking clinical trials to evaluate the handling and fate of nutrients by the fatty liver. We used the BreathID real-time breath test device in combination with a labeled orally-delivered fatty acid to demonstrate a decrease in the rate of fatty acid oxidation in subjects with NAFLD compared to controls. Similarly, we utilized a metabolomic approach to identify the response of NAFLD subjects to a standardized meal challenge. Finally, we are currently performing a clinical trial, aimed to elucidate the hepatic response to an oral carbohydrate load at the transcriptomic, lipidomic levels, and to identify prediction rules for response to a novel class of medications, GLP-1 receptor agonists.